A major tool in molecular biology and related fields is electrophoretic separation. In this process, mixtures of macromolecules, e.g., proteins, DNA, or RNA, are moved through a sieving medium, such as a gel, by an electric field. Electrophoretic separation enables qualitative analysis, separation, recovery, and purification of macromolecules.
Nucleic acid molecules have one negative charge for each nucleotide. As a result, the charge-to-mass ratio is constant for DNA of various lengths and an electric field alone would not perform separation of DNA. In a porous medium, however, larger DNA molecules have greater difficulty traversing the enmeshing medium and move slower than smaller DNA molecules, thereby causing separation of DNA molecules of varying length. This separation effect is size limited, however, and conventional gel and capillary electrophoresis separation of DNA molecules is generally limited to sizes smaller than 50 kilobase pairs (kbp).
U.S. Pat. No. 4,473,452 to Cantor teaches that electrophoresis of very large molecules can succeed by using alternating transverse electric fields. This method is known as pulsed-field gel electrophoresis (PFGE). It is generally believed that the chains of molecules presented to the PFGE gel are oriented and re-oriented by the fields, allowing the longer chains to traverse the gel medium without clogging the passages. In addition, the longer the chains are, the greater is the relaxation time involved in the changes of orientation. As a result, the smaller chains move into and through the gel matrix much more readily than the larger chains. As described by E. Lai, et al. (“Pulsed-field Gel Electrophoresis,” Bio Techniques, 7, pp. 34-42, 1989), the alternating electric fields can be applied in a number of orientations, giving rise to several variations of the PFGE method, e.g., field inversion gel electrophoresis (FIGE), clamped homogeneous electric fields (CHEF) electrophoresis, and pulsed homogeneous orthogonal field gel electrophoresis (PHOGE). The pulsed-field technique has also been applied to capillary electrophoresis for the separation of large DNA fragments as described by Kim and Morris (“Rapid Pulsed-field Capillary Electrophoretic Separation of Megabase Nucleic Acids,” Anal. Chem. 67, pp. 784-786, 1995).
One of the primary applications of PFGE is the molecular typing of bacteria. In this methodology, bacterial cells, imbedded in an agarose plug, are treated to lyse the cells and remove or destroy cellular proteins. The released chromosomal DNA is then treated with a restriction endonuclease enzyme that cleaves infrequently to cut the DNA into large fragments, typically between 50 and 800 kbp in size. These large DNA fragments are separated using PFGE to yield a DNA fingerprint which can be used to identify the bacterium at the species and sub-species level and to differentiate among related bacteria.
While extremely effective for the molecular typing of bacteria and other microbes, PFGE has the major disadvantage of an extremely time-consuming sample treatment process, wherein three to four days is not uncommon. One reason the sample treatment process is time-consuming is the need to protect the fragile DNA molecules from unintended mechanical breakage. Traditional methods for sample preparation in which the end product is DNA in solution are unsuitable for PFGE because large DNA molecules are susceptible to shearing forces leading to mechanical breakage. John Maule, “Pulsed-Field Gel Electrophoresis”, Molecular Biotechnology, Volume 9, 1998, pp. 107-126.
The method of U.S. Pat. No. 4,473,452 to Cantor avoids mechanical breakage of the long DNA molecules by incorporating the bacterial cells into molded inserts (also known as “plugs”), typically made of an agarose gel, and performing the sample treatment on the entrapped cells. This sample treatment includes lysis of the bacterial cells, enzymatic digestion of cellular proteins, and digestion of the DNA to produce fragments of various sizes using an appropriate restriction endonuclease enzyme. The treated plugs are then fitted into wells molded into the electrophoresis gel and PFGE is performed, resulting in a fingerprint pattern for the bacterium. By using the plugs, the DNA molecules can be extracted from the cell and digested in a controlled manner, without unwanted mechanical breakage of the DNA.
Molecular typing of bacterial cells by PFGE has typically been achieved using plugs. In this process, the cells or spheroplasts are suspended in gel (usually agarose) and then poured into molds to form the plugs. The sample treatment steps of lysis, deproteinization, and digestion are performed on the cells, embedded in the agarose plugs, as follows. First, the sample plugs are placed in a solution containing a lysing agent, e.g., lysozyme, and incubated at the appropriate temperature overnight. The lysing solution is then removed and the plugs are washed with buffer. A protease-containing solution, e.g., Proteinase K, is next added to digest proteins and the plugs are incubated overnight at the appropriate temperature. The next day, the protease solution is removed, and the plugs are washed several times with wash buffer. The plugs are washed with diluted wash buffer and then with the restriction enzyme buffer. A suitable restriction endonuclease-containing solution is then added to the plugs and the plugs are incubated overnight at the appropriate temperature. The next day, the restriction endonuclease solution is removed, and the plugs are washed with the wash buffer. A final wash is performed with the electrophoresis buffer, e.g., 0.5× tris-borate-EDTA (TBE) buffer. The plugs are then inserted into matching wells formed in a gel slab by a suitable comb and the pulsed-field electrophoretic separation is carried out. Care must be taken at every step to ensure that the plugs are not damaged in the process.
This sample plug treatment protocol avoids mechanical breakage in handling long and fragile DNA molecules. However, it makes sample treatment very tedious and time consuming, and the results are operator dependent. Sufficient time is required for diffusion of reagents into the agarose plugs. Moreover, the use of plugs makes automation of the sample preparation process very difficult, if not impossible. It is believed that there are no known reports of an automated sample treatment process for bacterial typing using PFGE.
Some practitioners have evolved one day protocols for PFGE employing plugs. But these protocols are often labor intensive and require highly skilled personnel. For example, Turabelidze, et al. (“Improved Pulsed-Field Gel Electrophoresis for Typing Vancomycin-Resistant Enterococci,” J. Clinical Microbiology November 2000, p. 4242-4245) describes a rapid protocol for sub-typing vancomycin-resistant enterococci in approximately one day. Gautom (“Rapid Pulsed-Field Gel Electrophoresis Protocol for Typing Of Escherichia coli 0157:H7 and Other Gram-Negative Organisms in 1 Day,” J. Clin. Microbiol. 35, November 1997, pp. 2977-2980) teaches a standardized protocol that is done in one day using bacterial cells directly from the culture plates, shortening cell lysis and deproteinization, using preheated buffer, and shorter restriction digestion times. In these more rapid methods, nevertheless, plugs are employed.
It has been reported in the CHEF-DR® II Pulsed-field Electrophoresis Systems Instruction Manual and Applications Guide from Bio-Rad Laboratories that liquid samples can be transferred and separated using PFGE when working with DNA in the size ranging from 50 kbp up to 200 kbp by taking special precautions not to mechanically break the DNA molecules. Specifically, the use of a pipet tip with a large opening is recommended. There is believed to be no known report of a non-shearing treatment and transfer of a liquid sample having DNA lengths greater than 200 kbp. For bacterial typing, the DNA fragments of interest often are greater than 200 kbp and typically range from 50 kbp to 1000 kbp, and are even greater than 1000 kbp in some instances. Therefore, for bacterial typing, sample plugs have been required to prevent mechanical breakage of the DNA molecules during sample treatment and gel loading.
There remains a need to reduce the sample treatment time required in the molecular typing of bacteria using PFGE. In addition, there is a need to eliminate the use of sample plugs in PFGE without causing unwanted mechanical breakage of the DNA molecules. There also is a need to treat and transfer bacterial samples for PFGE with a minimum degree of operator dependence by automating the sample treatment process.